1. Technical Field
The present invention relates to the field of animal health and in particular to attenuated pestiviruses such as classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV) or border disease virus (BDV).
2. Background Information
Pestiviruses are causative agents of economically important diseases of animals in many countries worldwide. Presently known virus isolates have been grouped into four different species which together form one genus within the family Flaviviridae.
I/II Bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and type 2 (BVDV-2) cause bovine viral diarrhea (BVD) and mucosal disease (MD) in cattle (Baker, 1987; Moennig and Plagemann, 1992; Thiel et al., 1996). The division of BVDV into 2 species is based on significant differences at the level of genomic sequences (summarized in Heinz et al., 2000) which are also obvious from limited cross neutralizing antibody reactions (Ridpath et al. 1994).
III Classical swine fever virus (CSFV), formerly named hog cholera virus, is responsible for classical swine fever (CSF) or hog cholera (HC) (Moennig and Plagemann, 1992; Thiel et al., 1996).
IV Border disease virus (BDV) is typically found in sheep and causes border disease (BD). After intrauterine infection of lambs with BDV persistently infected lambs can be born that are weak and show different abnormalities among which the ‘hairy shaker’ syndrome is best known (Moennig and Plagemann, 1992; Thiel et al., 1996).
Pestiviruses are small enveloped viruses with a single stranded RNA genome of positive polarity lacking both 5′ cap and 3′ poly(A) sequences. The viral genome codes for a polyprotein of about 4000 amino acids giving rise to final cleavage products by co- and posttranslational processing involving cellular and viral proteases. The viral proteins are arranged in the polyprotein in the order NH2-NPpro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Lindenbach and Rice, 2001). Protein C (=core- or capsidprotein) and the glycoproteins Erns, E1 and E2 represent structural components of the pestivirus virion as demonstrated for CSFV (Thiel et al., 1991). This also holds true for BVDV. E2 and to a lesser extent Erns were found to be targets for antibody neutralization (Donis et al., 1988; Paton et al., 1992; van Rijn et al., 1993; Weiland et al., 1990, 1992). Erns lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). The function of this enzymatic activity for the viral life cycle is presently unknown. The enzymatic activity depends on the presence of two stretches of amino acids conserved between the pestivirus Erns and different known RNases of plant and fungal origin. Both of these conserved sequences contain a histidine residue (Schneider et al., 1993). Inactivation of the RNase activity residing within the Erns results in an attenuated apathogenic pestivirus which is capable to be used as a modified live vaccine (WO 99/64604).
The pestivirus glycoprotein Erns is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. The most C-terminal cysteine residue forms the intermolecular disulfide bond between two Erns monomers, resulting in the Erns homodimer (Schneider et al., 1993). Recently it has been reported for CSFV that a substitution of the most C-terminal cysteine against serine results in a viable CSFV, which, however, lacks the ability to form any Erns homodimers (van Gennip et al., 2005).
Npro represents the first protein encoded by the long open reading frame in the pestivirus RNA. NPpro represents a nonstructural protein that has protease activity and cleaves itself of the nascent polyprotein (Stark et al., 1993; Wiskerchen et al., 1991) presumably already during translation. NPpro is a cysteine protease (Rümenapf et al., 1998) that is not essential for virus replication (Tratschin et al., 1998). Recently, it was shown that NPpro somehow interferes with the cellular antiviral defense so that it can be hypothesized to modulate the immune system within an infected host (Rüggli et al., 2003). Mayer and coworkers presented indications for an attenuation of CSFV in consequence of a deletion of the Npro gene (Mayer et al., 2004).
Present BVDV vaccines for the prevention and treatment of BVDV infections still have drawbacks (Oirschot et al. 1999). Vaccines against the classical BVDV-1 provide only partial protection from BVDV-2 infection, and vaccinated dams may produce calves that are persistently infected with virulent BVDV-2 (Bolin et al., 1991, Ridpath et al., 1994). This problem is probably due to the great antigenic diversity between type 1 and type 2 strains which is most pronounced in the glycoprotein E2, the major antigen for virus neutralization (Tijssen et al., 1996). Most monoclonal antibodies against type 1 strains fail to bind to type 2 viruses (Ridpath et al., 1994).
Vaccines comprising attenuated or killed viruses or viral proteins expressed in heterologous expression systems have been generated for CSFV and BVDV and are presently used. Conventional BVDV life vaccines are typically generated by cell culture passages resulting in viruses with attenuated virulence in the target species. The structural basis of the attenuation of BVDV used as life vaccines is not known. Therefore it is not possible to assess the molecular stability of the attenuation process. These vaccines, although attenuated, are most often associated with safety problems regarding use in breeding animals. The vaccine viruses may cross the placenta of pregnant animals, e.g. cows and lead to clinical manifestations in the fetus and/or the induction of persistently infected calves. The international patent application WO2005/111201 provides a new generation of a modified live pestivirus vaccine, which comprises a multiple modified pestivirus, having at least one mutation in the coding sequence for glycoprotein Erns and at least another mutation in the coding sequence for Npro, wherein said mutation in the coding sequence for glycoprotein Erns leads to inactivation of RNase activity residing in Erns and/or said mutation in the coding sequence for NPpro leads to inactivation of said NPpro.
However, in view of the importance of an effective and safe as well as detectable prophylaxis and treatment of pestiviral infections, there is a strong need for attenuated pestiviruses, such as BVDV, with a high potential for induction of immunity as well as a defined basis of attenuation which can also be distinguished from pathogenic pestiviruses, such as BVDV, as well as compositions and vaccines comprising said attenuated pestiviruses, such as BVDV.
Therefore, the technical problem underlying the present invention is to provide new attenuated pestiviruses, preferably an attenuated BVDV for use as live attenuated vaccines. Such improved attenuated pestivirus, preferably BVDV, should especially (i) not cross the placenta themselves and (ii) induce an immunity that prevents viral transmission across the placenta and thereby prevents pregnancy problems like abortion of the fetus or birth of persistently calves from infected host animals in the case of BVDV infection.
All subsequent Sequences are depicting the deleted regions with dashes (_d), which are also numbered, whereas the sequences in the sequence listing attached hereto are continuously numbered without the deleted regions or amino acid codons.    SEQ ID NO:1 (CSFV_wt) amino acid sequence of CSFV wildtype    SEQ ID NO:2 (CSFV_d) amino acid sequence of CSFV comprising a deletion of cystein at amino acid position 438 as compared to SEQ ID NO:1    SEQ ID NO:3 (CSFV_S) amino acid sequence of CSFV comprising a cystein/serine substitution at amino acid position 438 as compared to SEQ ID NO:1    SEQ ID NO:4 (BVDV_Ke9_wt) amino acid sequence of BVDV type 1 wildtype    SEQ ID NO:5 (BVDV_Ke9_d) amino acid sequence of BVDV type 1 comprising a deletion of cystein at amino acid position 441 as compared to SEQ ID NO:4    SEQ ID NO:6 (BVDV_Ke9_S) amino acid sequence of BVDV type 1 comprising a cystein/serine substitution at amino acid position 441 as compared to SEQ ID NO:4    SEQ ID NO:7 (BVDV_NY_wt) amino acid sequence of BVDV type 2 wildtype    SEQ ID NO:8 (BVDV_NY_d) amino acid sequence of BVDV type 2 comprising a deletion of cystein at amino acid position 441 as compared to SEQ ID NO:7    SEQ ID NO:9 (BVDV_NY_S) amino acid sequence of BVDV type 2 comprising a cystein/serine substitution at amino acid position 441 as compared to SEQ ID NO:7    SEQ ID NO:10 (BDV_x818_wt) amino acid sequence of BDV wildtype    SEQ ID NO:11 (BDV_x818_d) amino acid sequence of BDV comprising a deletion of cystein at amino acid position 439 as compared to SEQ ID NO:10    SEQ ID NO:12 (BDV_x818_S) amino acid sequence of BDV comprising a cystein/serine substitution at amino acid position 439 as compared to SEQ ID NO:10